Jurášek Michal, Göselová Sandra, Mikšátková Petra, Holubová Barbora, Vyšatová Eva, Kuchař Martin, Fukal Ladislav, Lapčík Oldřich, Drašar Pavel
University of Chemistry and Technology Prague, Prague, Czech Republic.
Czech Agriculture and Food Inspection Authority, Prague, Czech Republic.
Drug Test Anal. 2017 Apr;9(4):553-560. doi: 10.1002/dta.2005. Epub 2016 Jul 1.
Avidin-biotin technology was used for the implementation of an enzyme-linked immunosorbent assay (AB-ELISA) as a sensitive method for the detection of anabolic androgenic steroids (AAS) present in dietary supplements. Using click chemistry, novel haptens (linker-optimized biotinylated nandrolone (NT) and testosterone (T) at positions C-3 and C-17, respectively) were designed and synthesized to be then applied as four different immobilized competitors in a proposed set of four indirect competitive AB-ELISAs. Four rabbit polyclonal antibodies of various specificities were prepared using four different immunogens synthesized from C-3 and C-17 carboxymethyloxime and hemisuccinate derivatives of NT and T, respectively. Assembled AB-ELISAs were characterized to establish method parameters such as a half-maximum inhibition concentration (0.18-12.99 ng/mL), limit of detection (0.004-0.032 ng/mL) and linear working range (the best with 0.02-1.38 ng/mL). The stability of the set simulating storage in different conditions was demonstrated. Cross reactivity (CR) was tested for 59 steroids including both endogenous and synthetic analogues in four assembled AB-systems. The focus was placed on the practical use of the method in detection of various AAS in 49 samples of counterfeit dietary supplements. The concordance between ultra high performance liquid chromatography-mass spectrometry (UHPLC-MS) and the CR corrected data from AB-ELISA indicated the potential of this method even to quantification of T propionate, NT phenyl propionate, and NT decanoate in such a complex matter. Copyright © 2016 John Wiley & Sons, Ltd.
抗生物素蛋白-生物素技术被用于实施酶联免疫吸附测定（AB-ELISA），作为检测膳食补充剂中存在的合成代谢雄激素类固醇（AAS）的一种灵敏方法。利用点击化学，设计并合成了新型半抗原（分别在C-3和C-17位连接优化的生物素化诺龙（NT）和睾酮（T）），然后将其用作一组四种间接竞争AB-ELISA中的四种不同固定化竞争物。使用分别由NT和T的C-3和C-17羧甲基肟及半琥珀酸衍生物合成的四种不同免疫原制备了四种具有不同特异性的兔多克隆抗体。对组装好的AB-ELISA进行表征以确定方法参数，如半数抑制浓度（0.18 - 12.99 ng/mL）、检测限（0.004 - 0.032 ng/mL）和线性工作范围（最佳为0.02 - 1.38 ng/mL）。证明了该组在不同条件下模拟储存的稳定性。在四个组装好的AB系统中，对包括内源性和合成类似物在内的59种类固醇进行了交叉反应性（CR）测试。重点在于该方法在检测49份假冒膳食补充剂样品中各种AAS的实际应用。超高效液相色谱-质谱联用（UHPLC-MS）与AB-ELISA的CR校正数据之间的一致性表明了该方法甚至在如此复杂物质中对丙酸睾酮、苯丙酸诺龙和癸酸诺龙进行定量的潜力。版权所有© 2016约翰威立父子有限公司。
